The present invention relates to therapeutic compositions and methods for the treatment or diagnosis of diseases or conditions related to ICAM-1 levels, such as transplant rejection, cancer, rheumatoid arthritis, asthma, reperfusion injury, and inflammatory or autoimmune disorders. For example, such treatments will be useful for transplant rejection, myocardial ischemia, stroke, psoriasis, and Kawasaki disease.
The following is a brief description of the physiological role of ICAM-1. The discussion is not meant to be complete and is provided only for understanding of the invention that follows. This summary is not an admission that any of the work described below is prior art to the claimed invention.
Intercellular adhesion molecule-1 (ICAM-1) is a cell surface protein whose expression is induced by inflammatory mediators. ICAM-1 is required for adhesion of leukocytes to endothelial cells and for several immunological functions including antigen presentation, immunoglobulin production and cytotoxic cell activity. Blocking ICAM-1 function prevents immune cell recognition and activity during transplant rejection and in animal models of rheumatoid arthritis, asthma and reperfusion injury.
Cell-cell adhesion plays a pivotal role in inflammatory and immune responses (Springer et al., 1987 Ann. Rev. Immunol. 5, 223-252). Cell adhesion is required for leukocytes to bind to and migrate through vascular endothelial cells. In addition, cell-cell adhesion is required for antigen presentation to T cells, for B cell induction by T cells, as well as for the cytotoxicity activity of T cells, NK cells, monocytes or granulocytes. Intercellular adhesion molecule-1 (ICAM-1) is a 110 kilodalton member of the immunoglobulin superfamily that is involved in all of these cell-cell interactions (Simmons et al., 1988 Nature (London) 331, 624-627).
ICAM-1 is expressed on only a limited number of cells and at low levels in the absence of stimulation (Dustin et al., 1986 J. Immunol. 137, 245-254). Upon treatment with a number of inflammatory mediators (lipopolysaccharide, xcex3-interferon, tumor necrosis factor-xcex1, or interleukin-1), a variety of cell types (endothelial, epithelial, fibroblastic and hematopoietic cells) in a variety of tissues express high levels of ICAM-1 on their surface (Sringer et. al. supra; Dustin et al., supra; and Rothlein et al., 1988 J. Immunol. 141, 1665-1669). Induction occurs via increased transcription of ICAM-1 mRNA (Simmons et al., supra). Elevated expression is detectable after 4 hours and peaks after 16-24 hours of induction.
ICAM-1 induction is critical for a number of inflammatory and immune responses. In vitro, antibodies to ICAM-1 block adhesion of leukocytes to cytokine-activated endothelial cells (Boyd, 1988 Proc. Natl. Acad. Sci. USA 85, 3095-3099; Dustin and Springer, 1988 J. Cell Biol. 107, 321-331). Thus, ICAM-1 expression may be required for the extravasation of immune cells to sites of inflammation. Antibodies to ICAM-1 also block T cell killing, mixed lymphocyte reactions, and T dell-mediated B cell differentiation, suggesting that ICAM-1 is required for these cognate cell interactions (Boyd et al., supra). The importance of ICAM-1 in antigen presentation is underscored by the inability of ICAM-1 defective murine B cell mutants to stimulate antigen-dependent T cell proliferation (Dang et al., 1990 J. Immunol. 144, 4082-4091). Conversely, murine L cells require transfection with human ICAM-1 in addition to HLA-DR in order to present antigen to human T cells (Altmann et al., 1989 Nature (London) 338, 512-514). In summary, evidence in vitro indicates that ICAM-1 is required for cell-cell interactions critical to inflammatory responses, cellular immune responses, and humoral antibody responses.
This invention relates to ribozymes, or enzymatic RNA molecules, directed to cleave mRNA species encoding ICAM-1. In particular, applicant describes the selection and function of ribozymes capable of cleaving this RNA and their use to reduce levels of ICAM-1 in various tissues to treat the diseases discussed herein. Such ribozymes are also useful for diagnostic uses.
Ribozymes that cleave ICAM-1 mRNA represent a novel therapeutic approach to inflammatory or autoimmune disorders. ICAM-1 function can be blocked therapeutically using monoclonal antibodies. Ribozymes have the advantage of being generally immunologically inert, whereas significant neutralizing anti-IgG responses can be observed with some monoclonal antibody treatments. Antisense DNA molecules have been described that block ICAM-1 expression (Chiang et al., 1991 J. Biol. Chem. 266, 18162-18171). However, ribozymes may show greater perdurance or lower effective doses than antisense molecules due to their catalytic properties and their inherent secondary and tertiary structures. Such ribozymes, with their catalytic activity and increased site specificity (as described below), represent more potent and safe therapeutic molecules than antisense oligonucleotides.
Applicant indicates that these ribozymes are able to inhibit expression of ICAM-1 and that the catalytic activity of the ribozymes is required for their inhibitory effect. Those of ordinary skill in the art, will find that it is clear from the examples described that other ribozymes that cleave target ICAM-1 encoding mRNAs may be readily designed and are within the invention.
Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf et al., 1992 Proc. Natl. Acad. Sci. USA, 89, 7305-7309). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.
In preferred embodiments of this invention, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence). Examples of such hammerhead motifs are described by Rossi et al., 1992 Aids Research and Human Retroviruses, 8, 183, of hairpin motifs by Hampel et al., xe2x80x9cRNA Catalyst for Cleaving Specific RNA Sequences,xe2x80x9d filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, 1989 Biochemistry, 28, 4929 and Hampel et al., 1990 Nucleic Acids Research 18, 299, and an example of the hepatitis delta virus motif is described by Perrotta and Been, 1992, Biochemistry 31, 16, of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849, of the Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799) and of the Group I intron by Cech et al., U.S. Pat. No. 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
The invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target ICAM-1 encoding mRNA such that specific treatment of a disease; or condition can be provided with either one or several enzymatic nucleic acids. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required. Alternatively, the ribozymes can be expressed from DNA vectors that are delivered to specific cells.
Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small enzymatic nucleic acid motifs. (e.g., of the hammerhead or the hairpin structure) are used for exogenous delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure. However, these catalytic RNA molecules can also be expressed within cells from eukaryotic promoters,(e.g., Scanlon et al., 1991 Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992 Antisense Res. Dev. 2, 3-15; Dropulic et al., 1992 J Virol. 66, 1432-41; Weerasinghe et al., 1991 J Virol. 65, 5531-5534; Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA, 89, 10802-10806; Chen et al., 1992 Nucleic Acids Res., 20, 4581-1589; Sarver et al., 1990 Science, 247, 1222-1225). Those skilled in the art realize that any ribozyme can be expressed in eukaryotic cells from the appropriate DNA vector. The activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Draper et al., PCT WO93/23569, and Sullivan et al., PCT WO94/02595, both hereby incorporated in their totality by reference herein; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991 Nucleic Acids Res., 19, 5125-5130; Ventura et al., 1993 Nucleic Acids Res., 21, 3249-55).
Thus, in a first aspect, the invention features ribozymes that inhibit ICAM-1 production. These chemically or enzymatically synthesized RNA molecules contain substrate binding domains that bind to accessible regions of their target mRNAs. The RNA molecules also contain domains that catalyze the cleavage of RNA. The RNA molecules are preferably ribozymes of the hammerhead or hairpin motif. Upon binding, the ribozymes cleave the target ICAM-1 encoding mRNAs, preventing translation and protein accumulation. In the absence of the expression of the target gene, a therapeutic effect may be observed.
By xe2x80x9cinhibitxe2x80x9d is meant that the activity or level of ICAM-1 encoding mRNA is reduced below that observed in the absense of the ribozyme, and preferably is below that level observed in the presence of an inactive RNA molecule able to bind to the same site on the mRNA, but unable to cleave that RNA.
Such ribozymes are useful for the prevention of the diseases and conditions discussed above, and any other diseases or conditions that are related to the level of ICAM-1 activity in a cell or tissue. By xe2x80x9crelatedxe2x80x9d is meant that the inhibition of ICAM-1 mRNA and thus reduction in the level of ICAM-1 will relieve to some extent the symptoms of the disease or condition.
Ribozymes are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells. The RNA or RNA complexes can be locally administered to relevant tissues or cells ex vivo or in vivo by injection or through the use of a catheter, infusion pump or stent, with or without their incorporation in biopolymers. In preferred embodiments, the ribozymes have binding arms which are complementary to the sequences in Tables II, III, VI-IX. Examples of such ribozymes are shown in Tables IV-VIII and X. Examples of such ribozymes consist essentially of sequences defined in these Tables. By xe2x80x9cconsists essentially ofxe2x80x9d is meant that the active ribozyme contains an enzymatic center equivalent to those in the examples, and binding arms able to bind mRNA such that cleavage at the target site occurs. Other sequences may be present which do not interfere with such cleavage.
In another aspect of the invention, ribozymes that cleave target molecules and inhibit ICAM-1 activity are expressed from transcription units inserted into DNA, RNA, or viral vectors. Preferably, the recombinant vectors capable of expressing the ribozymes are locally delivered as described above, and transiently persist in target cells. Once expressed, the ribozymes cleave the target mRNA. The recombinant vectors are preferably DNA plasmids or adenovirus vectors. However, other mammalian cell vectors that direct the expression of RNA may be used for this purpose.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.